Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme

Michiko Takahashi; Hajime Takahashi; Yumiko Okakura; Masahiro Ichikawa; Takashi Kuda; Bon Kimura

doi:10.1371/journal.pone.0237888

Peer Review History

Original SubmissionMay 18, 2020
16 Jul 2020 Decision Letter - Oscar Millet, Editor PONE-D-20-14783 Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme PLOS ONE Dear Dr. Takahashi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers are quite positive about the work but they both raised a number of issues that have to be addressed before the manuscript becomes acceptable. Please submit your revised manuscript by Aug 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Oscar Millet Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Financial Disclosure section: "This work was supported by Grant-in-Aid for JSPS Fellows Grant Number 17J05482, and Grant-in-Aid for Scientific Research (B) Grant Number 17H03872." We note that one or more of the authors are employed by a commercial company: "Kewpie Corporation," a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In previous work (10.1038/srep11819), the authors demonstrated that thermal/structural denaturation activates residues Lz23-57 (Lzm 5-39?) of egg white lysozyme in order to inactivate norovirus, a food contaminant. The present work explores the optimal inactivating conditions of denatured lysozyme. Optimal and complete inactivation of the glycoside hydrolase was found by raising the pH of the egg-white lysozyme solution above 6.5 prior to irreversible thermal denaturation, which reduced alpha helical content in CD spectra. Hydrophobic residues spanning the region 5-39 in the lysozyme sequence were posited to contribute to inactivation based on data for three mutant constructs. These findings are of relevance to the use of disinfectants in industrial food production. 1) It is not clear why the residues are numbered differently in the two reports by the authors (Lz 23-57 vs. Lzm 5-39). 2) The rationale for why/how the authors chose the three peptide variants is not entirely clear. The overall basic/positive peptide (6 basic, 3 acidic groups, pI = 11) was made to be either R1: neutral and amphiphilic (H->E and K->E mutations); R2: hydrophilic instead of hydrophobic (Asn instead of one Trp and two Phe); or R3: polar and more positive (3 acidic sidechains replaced with Asn). For instance, unlike Phe, tryptophan is a polar residue. Inactivation was lost with R1 and R2 and reduced in R3. It seems an oversimplification to say that the results show that 5-39 “hydrophobicity” is responsible for inactivation. Please describe the mutations/variants in the Abstract. 3) Line 310: it is presumably the “surface-exposed” hydrophobic and thiol content that increases with denaturation. The logic of mere “increased hydrophobicity” particularly breaks down in Lines 336-342. Lzm 1-35 and Lzm 5-39 were equivalent, so it seems that structural/surface changes upon denaturation are the culprit rather than the mere presence of hydrophobic residues (such as VFG). The authors propose that the exposed hydrophobic sequences bind the norovirus capsid rather than lysozyme having enzymatic activity, similar to the mechanism of gram-negative activation. 4) The readability of the manuscript could be improved by reorganizing passages. Methodological details are included in the Figure captions. The paragraph in the Discussion describes the effect of the mutant variants on protein charge, but this rationale is not explained in the earlier Introduction/Methods/Results sections. The Discussion would benefit from breaking up into subheadings/sections. Reviewer #2: In this manuscript, Takahashi et al attempt to delineate the mechanism of inactivation of murine norovirus by heat-denatured hen egg white lysozyme (DL). They show that increasing the pH to 6.5-8.5 before thermal denaturation increase the effectiveness of DL. It is known from an earlier study that residues 5-39 was the most effective at virus inhibition. Here the authors introduce mutations to modify net charge and hydrophobicity and conclude that both hydrophobicity as well positive charge of this peptide contribute to viral inhibition. It is also interesting that a simple clustering of hydrophobic residues in the primary sequence is not sufficient, rather the results with peptide 88-125 in this paper along with alpha-lactalbumin from a previous study (Takahashi et al, Scientific Reports, 2015) suggest that exposure of hydrophobic and charged residues in a particular conformation must be responsible for this. Of course, it is hard to imagine a ‘conformation’ in a heat denatured protein, and hence further studies are needed. Overall, the results presented in the paper are interesting, clear and understandable, and backed with evidence. As such it merits publication in Plos One, however a few issues need to be addressed before acceptance. 1. It would be good to include a hydropathy plot for each peptide along with the mutant variants. 2. Does the 5-39 peptide become totally unstructured in the presence of some denaturant? It is easy to get that information from a CD spectrum in the presence of some urea. This experiment will confirm that even the heat denatured peptide has some residual structure. Also, can you obtain CD spectrum of the heat denatured peptide in multiple batches and overlay? This will also make sure that this residual conformation is consistent every time you denature different batches. 3. You mention that increase in thiol content upon denaturation might be due to SS bonds breaking during heating. While this is possible, it would be great to quantify how many actually break (using Ellman’s reagent). This should be straight forward to do. 4. The organization of the manuscript needs to be changed. For example, there are several details about peptide design, and interpretation which are in the Methods and Discussion, while ideally, they should all be part of the Results. It is very difficult to go back and forth while reading. Methods should not contain the logic behind doing an experiment, rather should only focus on the technical details. Results of hydropathy index calculation are all included in Discussion, which again should move to Results. 5. The word ‘Denatured’ in keywords is misspelt. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Ron Hills Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0237888.r001
Revision 1
31 Jul 2020 Author Response Reponses to reviewer #1 1) It is not clear why the residues are numbered differently in the two reports by the authors (Lz 23-57 vs. Lzm 5-39). Thank you for your comments and suggestions. We found them quite useful as we approached our revision. - Sorry for confusing. In the previous report (Takahashi et al., 2015. Sci. Rep. 5: 11819), the reference sequence of lysozyme (GenBank ID: AAL69327) is a chicken lysozyme chromatin domain containing a lysozyme sequence at residues 19-145 (Chong et al., 2002. Nucleic Acids Res. 30:463-467). In this sequence, residues of 23-57 correspond to Lzm 5-39 of the lysozyme sequence (PDB ID: 6BO2). 2) The rationale for why/how the authors chose the three peptide variants is not entirely clear. The overall basic/positive peptide (6 basic, 3 acidic groups, pI = 11) was made to be either R1: neutral and amphiphilic (H->E and K->E mutations); R2: hydrophilic instead of hydrophobic (Asn instead of one Trp and two Phe); or R3: polar and more positive (3 acidic sidechains replaced with Asn). For instance, unlike Phe, tryptophan is a polar residue. Inactivation was lost with R1 and R2 and reduced in R3. It seems an oversimplification to say that the results show that 5-39 “hydrophobicity” is responsible for inactivation. Please describe the mutations/variants in the Abstract. - As you pointed out, the description about the background of peptide-design was not sufficient in the manuscript. - In the R1 variant, in order to make the number of acidic and basic amino acids the same, 2 out of 6 basic amino acids were replaced with acidic amino acids. Here, the hydropathy index was calculated to confirm that the hydropathy was almost the same as before replacement. - The R2 variant was aimed to change only the hydrophobicity without changing the charge. Lzm 5-39 originally had a large number of hydrophobic amino acids, and the replacement of all of the residues might prevent peptide-synthesis. Therefore, we decided to replace only the aromatic amino acids, which has a particularly high degree of hydrophobicity. - In the R3 variant, all the acidic amino acids were replaced with uncharged amino acids in order to shift the basic side. - The background of peptide design was added into the revised manuscript (P. 15, L. 253-268 in Revised Manuscript with Track Change). We also revised abstract section to describe the peptide mutation/variants (P. 2, L. 31-37 in Revised Manuscript with Track Change). - As you commented, it cannot be concluded that only hydrophobicity contributes to virus-inactivating effect from the Fig. 4. Further study is needed to elucidate which of the characters, positive charge and hydrophobicity, dominates the virus-inactivation mechanism. It has been described in the manuscript (P. 22, L. 379-382 in Revised Manuscript with Track Change). 3) Line 310: it is presumably the “surface-exposed” hydrophobic and thiol content that increases with denaturation. The logic of mere “increased hydrophobicity” particularly breaks down in Lines 336-342. Lzm 1-35 and Lzm 5-39 were equivalent, so it seems that structural/surface changes upon denaturation are the culprit rather than the mere presence of hydrophobic residues (such as VFG). The authors propose that the exposed hydrophobic sequences bind the norovirus capsid rather than lysozyme having enzymatic activity, similar to the mechanism of gram-negative activation. - Fig. 4 implies that the surface-exposed hydrophobicity is important for virus inactivation, rather than the mere hydrophobicity of heat-denatured lysozyme as your comment. On the other hand, since it could not be asserted that “surface-exposed hydrophobicity is important” with Fig. 2 only. In response to the suggestion, we added sentence “” after the consideration of Fig. 4 (P. 20 L. 345-347 in Revised Manuscript with Track Change). 4) The readability of the manuscript could be improved by reorganizing passages. Methodological details are included in the Figure captions. The paragraph in the Discussion describes the effect of the mutant variants on protein charge, but this rationale is not explained in the earlier Introduction/Methods/Results sections. The Discussion would benefit from breaking up into subheadings/sections. - The structure of paragraph was reorganized as suggested by both reviewer #1 and reviewer #2. The description of mutant peptides, hydropathy index calculation, and hydropathy plot have been moved to the results section based on the proposal of reviewer #2 (P. 15 L. 253-268 in Revised Manuscript with Track Change). Responses to reviewer #2 1. It would be good to include a hydropathy plot for each peptide along with the mutant variants. We appreciate your comments and suggestions, which have helped us significantly improve the paper. We tried to be responsive to your concerns. - Hydropathy plot for each peptide was added in this revision as suggested (S Fig. 1). 2. Does the 5-39 peptide become totally unstructured in the presence of some denaturant? It is easy to get that information from a CD spectrum in the presence of some urea. This experiment will confirm that even the heat denatured peptide has some residual structure. Also, can you obtain CD spectrum of the heat denatured peptide in multiple batches and overlay? This will also make sure that this residual conformation is consistent every time you denature different batches. 3. You mention that increase in thiol content upon denaturation might be due to SS bonds breaking during heating. While this is possible, it would be great to quantify how many actually break (using Ellman’s reagent). This should be straight forward to do. - As your suggestion, acquiring CD spectrum in the presence of some urea and quantifying SS bonds could further enhance the manuscript. However, unfortunately we are unable to perform these additional experiments due to the current COVID-19 pandemic. 4. The organization of the manuscript needs to be changed. For example, there are several details about peptide design, and interpretation which are in the Methods and Discussion, while ideally, they should all be part of the Results. It is very difficult to go back and forth while reading. Methods should not contain the logic behind doing an experiment, rather should only focus on the technical details. Results of hydropathy index calculation are all included in Discussion, which again should move to Results. - We reorganized manuscript as your suggestion. In particular, some descriptions about peptide design, hydropathy index calculation, and hydropathy plot were moved to in the results section (P. 15, L. 253-268 in the Revised Manuscript with Track Change). 5. The word ‘Denatured’ in keywords is misspelt. - “Denatured” in keywords was corrected in this revision. Again, thank you for taking the time and energy to help us improve the paper. We are hopeful that our revision helps to improve your opinion of work. Attachments Attachment Submitted filename: Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0237888.r002
5 Aug 2020 Decision Letter - Oscar Millet, Editor Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme PONE-D-20-14783R1 Dear Dr. Takahashi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Oscar Millet Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0237888.r003
Formally Accepted
10 Aug 2020 Acceptance Letter - Oscar Millet, Editor PONE-D-20-14783R1 Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme Dear Dr. Takahashi: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Oscar Millet Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0237888.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .